Method for accessing microbial diversity

ABSTRACT

A method of interfering with quorum sensing regulation of genes to promote cell growth is disclosed. The method of is aimed at accessing microbial biodiversity. The method involves obtaining an environmental sample comprising at least one novel (uncultivated in the laboratory) microorganism, contacting the environmental sample with an effective amount of an agent or combination of agents which interferes with the quorum sensing regulation of genes, growing the treated sample in a culture medium containing the quorum sensing signal disrupting agent or agents, and analyzing the colonies of microorganisms grown to demonstrate genetic novelty.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to methods for accessing microbial diversitythrough disruption of microbial quorum sensing systems. In particular,this invention enables the isolation of novel microorganisms bydis-enabling quorum sensing systems that are used to maintain microbialcell density at a low level.

2. Description of Related Art

With recent developments in PCR technology and comparative microbialgenome sequencing, it has been demonstrated in many environments thatthe number of microorganisms that have been cultured represents only apercentage of those present in a particular environment. It has beenestimated that only approximately 1-5% of existing microorganisms havebeen cultured in the laboratory.

The organisms which remain “uncultivated” represent a potentially largepool of genes comprising novel microbial diversity. Accessing thisdiversity would allow the identification for example of enzymesexhibiting novel or enhanced biocatalytic characteristics, novelcofactors, or other novel secondary metabolites such as pharmaceuticals,polymers or other chemicals. Many industrial processes utilize (or couldutilize) microbial processes or components thereof and could thusbenefit greatly from the isolation of novel microorganisms exhibitingunique characteristics. In addition, novel microorganisms responsiblefor disease states could be identified. Also, environmentalbioremediation could benefit greatly from the identification ofmicroorganisms exhibiting novel biodegradation or bioconversionprocesses.

Many companies have recently been formed to access microbial diversityby using recombinant techniques to circumvent the inability to culturemicroorganisms. These techniques are limited in that they can onlyaccess single genes or small clusters of genes encoding short metabolicor biosynthetic pathways. The ability to cultivate a microorganism inthe laboratory would provide a tremendous advantage.

In nature, bacteria communicate with one another in order to coordinatethe expression of specific genes in a cell density-dependent manner.This bacterial communication is called quorum sensing, and it allowsbacteria to control gene expression in response to the level of adiffusible signaling molecule called an autoinducer. The signalingmolecule binds to a receptor protein, which then activates geneexpression. Processes which are controlled by quorum sensing includevirulence, bioluminescence, biofilm formation, swarming, sporulation,conjugal transfer of plasmids, and development of competence.

Three main types of quorum sensing systems have been described inbacteria: Type 1, Type 2 and peptide-based. Type 1 quorum sensing hasthus far only been demonstrated in Gram negative microorganisms andutilizes acyl homoserine lactones as signaling molecules. Type 2 hasbeen demonstrated in both Gram positive and Gram negative microorganismsand is believed to utilize 4-hydroxy-5-methyl-2H-furan-3-one or4,5-dihydroxy-2-cyclopenten-1-one as the signaling molecule.Peptide-based quorum sensing systems have been demonstrated only in Grampositive microorganisms and rely on short peptides for gene activation.In addition, other chemical signals have been shown to be used forquorum sensing; these include gamma butyrolactone in Streptomyces sp.and 2-heptyl-3-hydroxy-4-quinolone in Pseudomonas aeruginosa.

Type 1 quorum sensing utilizes acyl homoserine lactones (AHSL) assignaling molecules. AHSL chemical signals consist of a lactone ringattached to an acyl chain by means of a peptide bond. The acyl chainlength is specific for a given microorganism or for an AHSL-mediatedprocess carried out by that microorganism. Some AHSLs contain a carbonylor hydroxyl group at the 3 position of the acyl chain (e.g.,3-oxo-hexanoyl homoserine lactone, 3-hydroxy-butanoyl homoserinelactone). The paradigm for Type 1 quorum sensing is the Vibrio fischeriluxI/luxR system. The luxI protein catalyzes the synthesis of theautoinducer 3-oxo-hexanoyl homoserine lactone (OHHL). As the celldensity increases the autoinducer accumulates and when a threshold levelis reached, the OHHL signal interacts with the luxR protein. TheluxR/OHHL complex binds to DNA at the lux box resulting in transcriptionof the bioluminescence genes. Other microorganisms exhibiting Type 1quorum sensing possess analogs of luxI and luxR.

WO 01/85664 is incorporated in its entirety for its description of Type2 quorum sensing. Biosynthesis of the Type 2 autoinducer is believed toproceed through progressive steps from methionine through S-adenosylmethionine to S-adenosyl homocysteine to S-ribosyl homocysteine to4-hydroxy-5-methyl-2H-furan-3-one or 4,5-dihydroxy-2-cyclopenten-1-one.Enzymes involved in the synthesis are believed to include methionineadenosyl transferase, methyl transferase, nucleosidase and the luxSprotein or its analogs, which synthesizes4-hydroxy-5-methyl-2H-furan-3-one or 4,5-dihydroxy-2-cyclopenten-1-onefrom its precursor. In Vibrio harveyi, the receptors for the Type 2autoinducer are luxP and luxPQ. When autoinducer concentrations reach athreshold level, the autoinducer interacts with the receptor and luxO isdephosphorylated (and inactivated), thereby preventing activation of arepressor and allowing luxR to activate transcription of the luxCDABEgenes.

Many Gram positive bacteria use secreted peptides as autoinducers.Generally, in peptide based quorum sensing systems, the peptide issecreted by an ATP-binding cassette (ABC) transporter. The concentrationof the autoinducer increases with cell density, and at a threshold leveltwo component sensor kinases detect the autoinducer. A phoshorylationcascade is initiated which results in phosphorylation of a cognateresponse regulator protein. The response regulator is thus activated,allowing it to bind DNA and affect transcription of the quorum-sensingregulated genes.

In nature microorganisms regulate microbial processes in response toenvironmental conditions. In environments, for example, where nutrientsare uniformly distributed, it is conceivable that microorganismsregulate cell division such that a high cell density is never achievedand cells remain dispersed; an example of such an environment is theocean. It is possible that microorganisms utilize quorum sensing tocontrol their cell division, and thus many microorganisms from theseenvironments would thus far have been uncultivable in the laboratory dueto quorum sensing. Therefore, if quorum sensing were disrupted ordis-enabled, novel microorganisms from these environments could becultivated in the laboratory.

BRIEF DESCRIPTION OF THE INVENTION

Some bacteria produce chemical signals that regulate their own celldensity. It has been suggested that quorum sensing signal molecules mayinhibit the growth of daughter cells of the bacteria producing thequorum sensing signal molecules thereby poising the cell population atlow density. Such a mechanism of sustaining a relatively low celldensity may also contribute to the difficulties experienced bymicrobiologists in trying to establish pure cultures of these bacteria.Removal of the signal, blocking its production or inhibiting theactivity of the signal may allow cell density to increase and therebyresult in a “colony” or cell mass that can be visualized.

The present invention relates to a method of interfering with quorumsensing in order to inhibit the mechanism that regulates cell populationgrowth. The method involves a) obtaining an environmental samplecomprising at least one novel (uncultivated in the laboratory)microorganism, b) contacting the environmental sample with an effectiveamount of an agent or combination of agents that at least one of whichinterferes with, disrupts, removes, or dis-enables one or more quorumsensing signals in the environmental sample, c) growing the treatedsample in a culture medium containing the quorum sensing disruptingagent or agents, and d) analyzing the colonies of microorganisms grownto demonstrate genetic novelty minimally at the species or subspecieslevel.

The invention allows for the capturing of previously unculturablemicrobial biodiversity.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to a method for accessing microbial diversity byinterfering with or disrupting quorum sensing signals that regulate thepopulation density of a microorganism by adding an agent or combinationof agents that interferes with, disrupts, removes, or dis-enables thequorum sensing chemical signal system that regulates the populationdensity of the microorganism. Where one of the words “interfering with,disrupting, removing, inhibiting or dis-enabling” appears in thisspecification the other words can be substituted.

The method of the present invention comprises: a) obtaining anenvironmental sample comprising at least one novel (uncultivated in thelaboratory) microorganism, b) contacting the environmental sample withan effective amount of an agent or combination of agents that at leastone of which interferes with, disrupts, removes, or dis-enables one ormore quorum sensing signals in the environmental sample, c) growing thetreated sample in a culture medium containing the quorum sensing signaldisrupting agent or agents, and d) analyzing the colonies ofmicroorganisms grown to demonstrate genetic novelty minimally at thespecies or subspecies level.

The environmental sample can be obtained from any source that isexpected to contain at least one novel (i.e., not yet cultured)microorganism. Sources of the environmental sample expected to containnovel microorganisms include any body of water including but not limitedto streams, rivers, lakes, ponds, swamps, oceans, and the like. Sourcesalso include soil samples from any variety of sources. Soil samples canbe taken from any type of forest, such as rain forest or deciduous. Inaddition, soil samples can be taken from grass lands, wet lands, tundra,dry arid regions, farm lands, the bottoms of lakes, rivers or oceans andthe like. Samples may also be taken from geological formations, orenvironments exposed to extremes of temperature or pressure, includingextraterrestrial environments. Examples of temperature extremes includebut are not limited to the Arctic and Antarctic, or hydrothermal ventsor hot springs. Samples may also be obtained from other microorganismsor multicellular organisms (including humans) containing microbialsymbionts or pathogens; samples may also consist of bodily fluids.Environmental samples may also include, for example, plant components,such as leaves or the rhizosphere. Environmental samples may alsoinclude samples from industrial processes.

The invention relates to a method for interfering with, disrupting,removing, inhibiting or dis-enabling the acyl homoserine lactone (AHSL)chemical signals (autoinducers) which facilitate Type 1 quorum sensingin many Gram negative bacteria. AHSL signals may be disrupted using anagent that a) opens the lactone ring, b) hydrolyzes the peptide bond, orc) modifies the acyl chain.

In one embodiment of the invention, the agent is an enzyme thatcatalyzes a reaction with the acyl homoserine lactone autoinducer.Examples of classes of enzymes include esterases, lipases, lactonases,proteases, peptidases, aminoacylases or carboxypeptidases; many enzymescomprising these classes are commercially available.

It has been demonstrated that enzymes can degrade AHSLs. Lactonase hasbeen shown to inactivate oxohexanoyl-, oxodecanoyl- andoxooctanoyl-homoserine lactones (Dong et al. PNAS USA 97:3526-331, 2000and Nature 411:813-817, 2001). Similarly, it has been demonstrated thata strain of Variovorax paradoxus can utilize several acyl homoserinelactones for growth; it is believed that the ring is enzymaticallycleaved allowing the acyl chain and lactone ring to be used as sourcesof energy and nitrogen, respectively (Leadbetter and Greenberg, J.Bacteriology, 182:6921-6926). In another embodiment, the agent is achemical other than an enzyme that catalyzes a reaction with theautoinducer molecule, such that the structure of the autoinducer ismodified and the autoinducer becomes non-functional. Addition of sodiumhydroxide or other base to raise the pH to greater than 8 is known tohydrolyze the lactone ring, thereby degrading the AHSL.

In one embodiment of the invention, the agent of the invention is achemical that inhibits biosynthesis of the acylhomoserine lactoneautoinducer, such as by inhibiting the luxI protein, an analog thereof,or a protein exhibiting a similar function. Examples of such an agentinclude cycloleucine or (2S,4S)-2-amino-4,5-epoxy pentanoic acid,inhibitors of S-adenosylmethionine synthesis. In another embodiment ofthe invention, the agent is a chemical that inhibits binding of the acylhomoserine lactone autoinducer to its receptor, thus blockingtranscription of quorum sensing regulated genes. An example of such achemical is an antibody that specifically binds to the receptor; theantibody may be polyclonal or monoclonal and can be prepared usingmethods that are well known in the art. An additional example of such achemical is an analog of the AHSL itself. Halogenated furanones from thered alga Delisea pulchra which inhibit binding of the AHSL to thereceptor that regulates swarming in Serratia liquefaciens are an exampleof an analog of an AHSL (Rasmussen, et al., Microbiology, 146:3237-3244,2000).

In another embodiment of the invention, the invention relates to amethod for interfering with, disrupting, removing, inhibiting ordis-enabling Type 2 quorum sensing. In one embodiment, the agent is anenzyme that catalyzes a reaction with the Type 2 quorum sensingautoinducer, 4-hydroxy-5-methyl-2H-furan-3-one,4,5-dihydroxy-2-cyclopenten-1-one or an analog. In another embodiment,the agent is a chemical that disrupts the Type 2 autoinducer.

In yet another embodiment, the agent is a chemical that inhibitsbiosynthesis of the Type 2 quorum sensing autoinducer. Agents inhibitingthe biosynthesis of the Type 2 autoinducer can modify the biosyntheticenzymes themselves. Alternatively the agent can be an analog of one ofthe biosynthetic precursors of one of the enzymes. For example, theagent can be an analog of methionine, S-adenosyl homocysteine, orS-ribosylhomocysteine, thus preventing binding of these molecules to theappropriate enzyme and biosynthesis of the autoinducer.

In another embodiment of the invention, the agent is a chemical thatinhibits binding of the Type 2 quorum sensing autoinducer to itsreceptor. The agent can be a chemical that modifies luxP or luxQ, orproteins that carry out similar functions in other organisms. Similarlyan agent can inhibit Type 2 quorum sensing by modifying luxO, luxR orthe repressor protein, or any of the proteins that carry out similarfunctions in other organisms. The agent can also bind to the autoinducerreceptor or other proteins involved in signal transduction between theautoinducer and the quorum sensing-controlled genes; an example is anantibody that binds to one of the proteins involved. In anotherembodiment, the agent can be an analog of the Type 2 autoinducermolecule, such as a modified furanone.

In another embodiment, the invention relates to a method of interferingwith, disrupting, removing, inhibiting or dis-enabling peptide-regulatedquorum sensing by Gram positive bacteria. Many Gram positive bacteriause secreted peptides as autoinducers. In one embodiment, quorum sensingby Gram positive bacteria is inhibited by an enzyme that catalyzes areaction with the peptide autoinducer. Examples of such enzymes includebut are not limited to proteases, peptidases and deaminases. In someGram positive organisms, such as Staphylococcus, the peptide contains athiolactone ring; these autoinducers may also be disrupted by an enzymecatalyzing a reaction with the thiol bond, such as a thiol reductase. Inanother embodiment of the invention, the agent is a chemical thatdisrupts the structure of the autoinducer peptide such as by modifyingcarboxyl or amide groups. In still another embodiment of the invention,the agent is an antibody that binds to the autoinducer peptide, thuspreventing binding of the peptide to its receptor protein. The antibodymay also bind an autoinducer propeptide, thus preventingpost-translational processing to the active autoinducer. Peptidemimetics, such as β-peptides, may also inhibit binding of a peptide toits receptor.

In another embodiment of the invention, the agent is a chemical thatinhibits the biosynthesis of the autoinducer peptide. The agent may, forexample, inhibit transcription of the peptide or its propeptide (in thecase of autoinducers that are post-translationally modified). The agentmay inhibit the cleavage of the autoinducer peptide from its propeptide.

In another embodiment, the agent is a chemical that inhibits the bindingof the peptide to its receptor protein. The agent may be a chemical orenzyme that modifies the receptor or binds to the receptor, therebyinactivating it; an example is an antibody specific for the receptorwhich disrupts binding of the autoinducer to the receptor. In anotherembodiment, the agent is an analog of the autoinducer peptide whichbinds to the receptor, thereby preventing binding of the autoinducer.Current Opinion in Microbiology 1999, 2:40-45, the entire contents ofwhich is herein incorporated by reference, describes how an autoinducerfor one bacterial species may act as an inhibitor for another. Thesepeptides can be used as agents of inhibition in the present invention.There are numerous other references citing inhibitors that a personskilled in the art would recognize as being useful in the presentinvention.

Other quorum sensing autoinducer molecules have been described, such asgamma-butyrolactone from Streptomyces and 2-heptyl-3-hydroxy-4-quinolonefrom Pseudomonas aeruginosa. It is likely that additional quorum sensingsystems have not yet been described. Using the methods described above,it would be possible for one skilled in the art to disrupt these quorumsensing systems in order to allow colony formation by organisms thatregulate cell density by using quorum sensing.

Any combination of the agents can be used to interfere with, disrupt,remove, or dis-enable or inhibit quorum sensing. For a nonlimitingexample, an agent for the Type 1 autoinducer, an agent for the Type 2autoinducer and an agent for the peptide autoinducer can be mixedtogether and used on a given sample.

The quorum sensing inhibiting agents of the present invention arepreferably soluble in water and may be applied or delivered with anacceptable carrier system. The composition may be applied or deliveredwith a suitable carrier system such that the agent may be dispersed ordissolved in a stable manner so that the agent, when it is administereddirectly or indirectly, is present in a form in which it is available ina particularly advantageous way.

Also, the separate agents of the present invention may be preblended oreach component may be added separately to the same environment accordingto a predetermined dosage for the purpose of achieving the desiredconcentration level of the treatment components and so long as thecomponents eventually come into intimate admixture with each other.

In another embodiment of the invention, the method of the inventioncomprises utilizing a physical device which acts to keep theconcentration of the autoinducer below the threshold concentrationrequired for the quorum sensing response. An example of the physicaldevice may be a membrane or other solid support to which microorganismscan attach. The membrane is then continuously washed with growth mediumto maintain the concentration of the diffusible autoinducer at a lowconcentration. The physical device may comprise a polymer or other solidsupport to which antibodies specific for the autoinducer or theautoinducer receptor protein are bound. The autoinducer is thusprevented from binding to its receptor and quorum sensing is inhibited.Any type of physical support can be used that would house the membraneor solid support contemplated by this invention. Examples of suchphysical devices may include, but are not limited to, ultrafiltration ordiafiltration units or chromatography columns which could be used tohouse the appropriate membrane or solid support.

The culture medium used for growth of the treated samples can be anymedium known to those skilled in the art. Preferably, the medium isderived from components comprising the environment from which the sampleis taken. For example, a growth medium to isolate marine microorganismspreferably contains marine salts. A medium for growth of soilmicroorganisms may be derived from soil extract. Media can besupplemented with appropriate components in order to isolatemicroorganisms growing on a unique substrate or exhibiting a novelcharacteristic. The solid support used to isolate individual coloniescan be agar, agar noble, Gel-Rite or any other solid support known tothose skilled in the art. Strategies for the isolation and growth ofmicroorganisms are described in part in The Prokaryotes (Martin Dworkin,ed., Springer-Verlag, NY, 1999).

In order to identify a microorganism as novel, the nucleic acid of themicroorganism must be sequenced and compared to databases of knownmicroorganisms. DNA sequencing can be done by a company or organizationspecializing in sequencing or can be done by the investigator usingmethods and equipment known in the art.

EXAMPLES Example 1

Growth and isolation of novel microorganisms: Utilization of esteraseSigma E0887 to degrade homoserine lactone (Type 1) autoinducer signals.

In this example water from a lake/reservoir is used as the source ofmicroorganisms, however any source that is expected to exhibit adiversity of novel (i.e., not yet cultured) microorganisms may be used.

Water samples (approximately 1-2 liters) are obtained at a depth of 0-2meters from Marsh Creek Lake (Downingtown, Pa.) and Spruce Run Reservoir(Clinton, N.J.); the samples are maintained at 4-20 C until used.

Agar media are prepared consisting of 1) filter-sterilized sample water,0.5% Difco Agar Noble (Becton Dickinson, Sparks, Md.), and 0.05% DifcoBacto Peptone and 2) filter-sterilized sample water, 0.5% Difco AgarNoble, and 0.50% Difco Bacto Peptone. Preferably the agar and peptoneare prepared at 4× concentration in sample water, sterilized and thendiluted with filter-sterilized sample water to the appropriate volume.When necessary, the media are supplemented with 200 U/ml offilter-sterilized esterase (Sigma #E0887) or 200 U/ml of boiled,filter-sterilized esterase (esterase is boiled for 10 min at 100 C andthen cooled) (200 U/ml=final concentration). In this example theesterase Sigma E0887 is used to degrade the signal, however otherenzymes or inhibitors of the Type 1 system may be used.

The agar media are poured into Petri plates and either 1) pour platesare prepared or 2) the agar is allowed to solidify and samples arespread on the agar surface.

Three sets of water samples are prepared: a) sample water plus E0887(E0887 is dissolved in deionized water and filter-sterilized; finalconcentration in sample water 200 U/ml), b) sample water plus boiledE0887 (dissolved in deionized water, filter-sterilized, boiled for 10min, and cooled; final concentration 200 U/ml), and c) sample water plusan amount of sterile, deionized water equal to that added to samples a)and b). The samples are then diluted in successive 1:10 steps to 1×10⁻⁴,and 0.1 ml aliquots of the dilutions are plated into (pour plates) or onto the surface of the agar media. The agar plates are then incubated forup to 10 days at 20-30 C, preferably in a humidity-controlled chamber toprevent evaporation. The number of aliquots at each dilution and foreach medium is determined by the number of colonies required to obtainstatistically relevant differences in colony-forming units (CFU) on theplates with and without E0887.

Colony formation during growth can be monitored with a dissectingmicroscope, or alternatively, an indicator dye such as2,3,5-triphenyltetrazolium chloride (TTC; 0.005%) may enable bettervisualization of the colonies.

After incubation, colonies are counted on/in the plates. The number ofCFU on the plates containing esterase E0887 should be significantlygreater than those on the plates lacking E0887. The plates containingboiled E0887 should allow for determination of those organisms thatutilize peptides/proteins for growth; the number of CFU on these platesmay be similar to that for “medium minus E0887” due to the peptone inthe medium.

Colonies from the “medium plus E0887” plates are suspended in sterilesample water and transferred to fresh medium+/−E0887. Those coloniesthat grow on “medium+E0887”, but not on “medium−E0887” are thensubmitted to Accugenix™/Acculab (Newark, Del.) (or a laboratory offeringsimilar services) for DNA sequencing to determine if the microorganismsbelong to new genera, species or subspecies (i.e., “novel”).

Example 2

Growth and isolation of novel microorganisms: Utilization ofL-selenomethionine or adenine to inhibit the Type 2 autoinducer signal.

In this example water from the Atlantic Ocean is used as the source ofmicroorganisms, however any source that is expected to exhibit adiversity of novel (i.e., not yet cultured) microorganisms may be used.

Water samples (approximately 1-2 liters) are obtained at a depth of 0-2meters from the Atlantic Ocean, 1 mile east of Ocean City, Md.; thesamples are maintained at 4 C until used.

Agar media are prepared consisting of 1) filter-sterilized sample water,0.5% Difco Agar Noble (Becton Dickinson, Sparks, Md.), and 0.05% DifcoBacto Peptone and 2) filter-sterilized sample water, 0.5% Difco AgarNoble, and 0.50% Difco Bacto Peptone. Preferably the agar and peptoneare prepared at 4× concentration in sample water, sterilized and thendiluted with filter-sterilized sample water to the appropriate volume.When necessary, the media are supplemented with 10 mM (finalconcentration) L-(+)-selenomethionine (Aldrich #473944) or 5 mM (finalconcentration) adenine (Sigma #A-8626) as inhibitors of the Type 2autoinducer system. Additional inhibitors of the Type 2 system may alsobe used.

The agar media are poured into Petri plates and either 1) pour platesare prepared or 2) the agar is allowed to solidify and samples arespread on the agar surface.

Two sets of water samples are prepared: a) sample water plusL-selenomethionine or adenine (dissolved in deionized water andfilter-sterilized; final concentration 10 mM or 5 mM, respectively), andb) sample water plus an amount of sterile, deionized water equal to thatadded with the inhibitor in sample a). The samples are then diluted insuccessive 1:10 steps to 1×10⁻⁴, and 0.1 ml aliquots of the dilutions toare plated into (pour plates) or on to the surface of the agar media.The agar plates are then incubated for up to 10 days at 10-18 C,preferably in a humidity-controlled chamber to prevent evaporation. Thenumber of aliquots at each dilution and for each medium is determined bythe number of colonies required to obtain statistically relevantdifferences in colony-forming units (CFU) on the plates with and withoutinhibitor.

Colony formation during growth can be monitored with a dissectingmicroscope, or alternatively, an indicator dye such as2,3,5-triphenyltetrazolium chloride (TTC; 0.005%) may enable bettervisualization of the colonies.

After incubation, colonies are counted on/in the plates. The number ofCFU on the plates containing inhibitor should be significantly greaterthan those on the plates lacking inhibitor.

Colonies from the “medium plus inhibitor” plates are suspended insterile sample water and transferred to fresh medium+/−inhibitor. Thosecolonies that grow on “medium+inhibitor”, but not on “medium−inhibitor”are then submitted to Accugenix™/Acculab (Newark, Del.) (or a laboratoryoffering similar services) for DNA sequencing to determine if themicroorganisms belong to new genera, species or subspecies (i.e.,“novel”).

Example 3

Growth and isolation of novel microorganisms: Utilization of the peptideAsp-Ile-Cys-Asn-Ala-Tyr-Phe to inhibit the Gram positive, peptide-basedsignaling system.

In this example soil is used as the source of microorganisms, howeverany source that is expected to exhibit a diversity of novel (i.e., notyet cultured) microorganisms may be used.

Soil samples (approximately 500 g) are obtained from the O_(h) and A_(h)horizons of a decidous forest; the samples are maintained at 4 C untilused.

Portions of the soil samples are washed 1:1 in tap water, filteredthrough Whatman #1 filter paper to obtain “soil water” andfilter-sterilized. Agar media are prepared consisting of 1)filter-sterilized soil water, 0.5% Difco Agar Noble (Becton Dickinson,Sparks, Md.), and 0.05% Difco Bacto Peptone and 2) filter-sterilizedsoil water, 0.5% Difco Agar Noble, and 0.50% Difco Bacto Peptone.Preferably the agar and peptone are prepared at 4× concentration in soilwater, sterilized and then diluted with filter-sterilized soil water tothe appropriate volume. When necessary, the media are supplemented withthe peptide Asp-Ile-Cys-Asn-Ala-Tyr-Phe (</=10 mM [final concentration])as an inhibitor of Gram positive quorum-sensing peptide(s).

The agar media are poured into Petri plates and either 1) pour platesare prepared or 2) the agar is allowed to solidify and samples arespread on the agar surface.

Portions of the soil samples are washed 1:1 in sterile tap water. Soilparticles are allowed to briefly settle, and two sets of water samplesare prepared: a) soil water plus filter-sterilizedAsp-Ile-Cys-Asn-Ala-Tyr-Phe (</=10 mM [final concentration]), and b)soil water plus an amount of sterile, deionized water equal to thatadded with the inhibitor in sample a). The samples are then diluted insuccessive 1:10 steps to 1×10⁻⁴, and 0.1 ml aliquots of the dilutionsare plated into (pour plates) or on to the surface of the agar media.The agar plates are then incubated for up to 10 days at 10-18 C,preferably in a humidity-controlled chamber to prevent evaporation. Thenumber of aliquots at each dilution and for each medium is determined bythe number of colonies required to obtain statistically relevantdifferences in colony-forming units (CFU) on the plates with and withoutpeptide inhibitor.

Colony formation during growth can be monitored with a dissectingmicroscope, or alternatively, an indicator dye such as2,3,5-triphenyltetrazolium chloride (TTC; 0.005%) may enable bettervisualization of the colonies.

After incubation, colonies are counted on/in the plates. The number ofCFU on the plates containing inhibitor should be significantly greaterthan those on the plates lacking inhibitor.

Colonies from the “medium plus inhibitor” plates are suspended insterile soil in water and transferred to fresh medium+/−inhibitor. Thosecolonies that grow on “medium+inhibitor”, but not on “medium−inhibitor”are then submitted to Accugenix™/Acculab (Newark, Del.) (or a laboratoryoffering similar services) for DNA sequencing to determine if themicroorganisms belong to new genera, species or subspecies (i.e.,“novel”).

Example 4

Growth and isolation of novel microorganisms: Utilization of a peptidaseto inhibit the Gram positive, peptide-based signaling system.

In this example soil is used as the source of microorganisms, howeverany source that is expected to exhibit a diversity of novel (i.e., notyet cultured) microorganisms may be used.

Soil samples (approximately 500 g) are obtained from the O_(h) and A_(h)horizons of a decidous forest; the samples are maintained at 4 C untilused.

Portions of the soil samples are washed 1:1 in tap water, filteredthrough Whatman #1 filter paper to obtain “soil water” andfilter-sterilized. Agar media are prepared consisting of 1)filter-sterilized soil water, 0.5% Difco Agar Noble (Becton Dickinson,Sparks, Md.), and 0.05% Difco Bacto Peptone and 2) filter-sterilizedsoil water, 0.5% Difco Agar Noble, and 0.50% Difco Bacto Peptone.Preferably the agar and peptone are prepared at 4× concentration in soilwater, sterilized and then diluted with filter-sterilized soil water tothe appropriate volume. When necessary, the media are supplemented witha peptidase, such as the Type IV bacterial protease from Streptomycesgriseus (Sigma #P6911) as an inhibitor of the Gram positivequorum-sensing peptide(s). The peptidase must demonstrate sufficientefficacy against the peptide at low temperature and low peptideconcentration, and the peptidase preparation must not contain microbialinhibitors. The concentration of peptidase used for inhibition isdetermined empirically with purified peptide.

The agar media are poured into Petri plates and either 1) pour platesare prepared or 2) the agar is allowed to solidify and samples arespread on the agar surface.

Portions of the soil samples are washed well 1:1 in sterile tap water.Soil particles are allowed to briefly settle, and three sets of watersamples are prepared: a) soil water plus filter-sterilized peptidase, b)soil water plus filter-sterilized, boiled peptidase (peptidase is boiledat 100 C and then cooled), and c) soil water plus an amount of sterile,deionized water equal to that added with the peptidase in samples a) andb). The samples are then diluted in successive 1:10 steps to 1×10⁻⁴, and0.1 ml aliquots of the dilutions are plated into (pour plates) or on tothe surface of the agar media. The agar plates are then incubated for upto 10 days at 10-18 C, preferably in a humidity-controlled chamber toprevent evaporation. The number of aliquots at each dilution and foreach medium is determined by the number of colonies required to obtainstatistically relevant differences in colony-forming units (CFU) on theplates with and without peptidase.

Colony formation during growth can be monitored with a dissectingmicroscope, or alternatively, an indicator dye such as2,3,5-triphenyltetrazolium chloride (TTC; 0.005%) may enable bettervisualization of the colonies.

After incubation, colonies are counted on/in the plates. The number ofCFU on the plates containing the peptidase should be significantlygreater than those on the plates lacking the peptidase.

Colonies from the “medium plus peptidase” plates are suspended insterile soil water and transferred to fresh medium+/−peptidase. Thosecolonies that grow on “medium+peptidase”, but not on “medium−peptidase”are then submitted to Accugenix™/Acculab (Newark, Del.) (or a laboratoryoffering similar services) for DNA sequencing to determine if themicroorganisms belong to new genera, species or subspecies (i.e.,“novel”).

Example 5

Growth and isolation of novel microorganisms: Utilization of antibodiesto inhibit the Gram positive, peptide-based signaling system.

In this example soil is used as the source of microorganisms, howeverany source that is expected to exhibit a diversity of novel (i.e., notyet cultured) microorganisms may be used.

Soil samples (approximately 500 g) are obtained from the O_(h) and A_(h)horizons of a decidous forest; the samples are maintained at 4 C untilused.

Antibodies to either the “autoinducer propeptide” or to the peptideitself (the autoinducer propeptide is the protein which is cleaved bybacteria to produce the autoinducer) are prepared using known methods.

Portions of the soil samples are washed 1:1 in tap water, filteredthrough Whatman #1 filter paper to obtain “soil water” andfilter-sterilized. Agar media are prepared consisting of 1)filter-sterilized soil water, 0.5% Difco Agar Noble (Becton Dickinson,Sparks, Md.), and 0.05% Difco Bacto Peptone and 2) filter-sterilizedsoil water, 0.5% Difco Agar Noble, and 0.50% Difco Bacto Peptone.Preferably the agar and peptone are prepared at 4× concentration in soilwater, sterilized and then diluted with filter-sterilized soil water tothe appropriate volume. When necessary, the media are supplemented withan antibody to the propeptide of the autoinducer or to the autoinducerpeptide. The antibody binds to the autoinducer as it is secreted fromthe cells or to the propeptide, thus deactivating it. The antibody mustdemonstrate sufficient affinity for the peptide or propeptide at lowtemperature and low peptide/propeptide concentration, and the antibodypreparation must not contain microbial inhibitors. The concentration ofantibody used for inhibition is determined empirically with purifiedpeptide or propeptide.

The agar media are poured into Petri plates and either 1) pour platesare prepared or 2) the agar is allowed to solidify and samples arespread on the agar surface.

Portions of the soil samples are washed well 1:1 in sterile tap water.Soil particles are allowed to briefly settle, and two sets of watersamples are prepared: a) soil water plus filter-sterilized antibody, andb) soil water plus an amount of sterile, deionized water equal to thatadded with the antibody in sample a). The samples are then diluted insuccessive 1:10 steps to 1×10⁻⁴, and 0.1 ml aliquots of the dilutionsare plated into (pour plates) or on to the surface of the agar media.The agar plates are then incubated for up to 10 days at 10-18 C,preferably in a humidity-controlled chamber to prevent evaporation. Thenumber of aliquots at each dilution and for each medium is determined bythe number of colonies required to obtain statistically relevantdifferences in colony-forming units (CFU) on the plates with and withoutantibody.

Colony formation during growth can be monitored with a dissectingmicroscope, or alternatively, an indicator dye such as2,3,5-triphenyltetrazolium chloride (TTC; 0.005%) may enable bettervisualization of the colonies.

After incubation, colonies are counted on/in the plates. The number ofCFU on the plates containing the antibody should be significantlygreater than those on the plates lacking the antibody.

Colonies from the “medium plus antibody” plates are suspended insterile, soil water and transferred to fresh medium+/−antibody. Thosecolonies that grow on “medium+antibody”, but not on “medium−antibody”are then submitted to Accugenix™/Acculab (Newark, Del.) (or a laboratoryoffering similar services) for DNA sequencing to determine if themicroorganisms belong to new genera, species or subspecies (i.e.,“novel”).

Example 6

Growth and isolation of novel microorganisms: utilization ofsolid-phase-bound antibodies to inhibit a peptide-regulated signalingsystem in organisms of the Domain Archaea.

Antibodies to the autoinducer peptide are generated using methods knownin the art. The antibodies are then bound to NHS-activated Sepharose(Amersham Pharmacia Biotech) via primary amino groups according toprocedures developed by the manufacturer. A column is preparedcontaining the antibody-bound Sepharose. A sample is obtained from thesediment in a swamp. An anaerobic medium is prepared which consists ofswamp water supplemented with 0.05% peptone, antibiotics (penicillin andtetracycline) and titanium citrate as the reductant. The swamp sample isadded to the Sepharose column and organisms are allowed to bind for onehour. The medium is then continuously flushed over the column for up to5 days. In this manner, the autoinducer will be removed by the antibody,contaminating microorganisms of the Domain Bacteria will be killedthrough inclusion of the antibiotics, and thus the novel Archaea will beselected for. Microorganisms attached to beads can then be plated ontoanaerobic swamp water medium containing 0.5% Agar Noble in order toisolate the novel Archaea. Microorganisms from colonies are sequenced todetermine their novelty as described in Example 1.

Example 7

Growth and isolation of novel microorganisms: utilization of acontinuous flow-device to inhibit quorum sensing and isolate novel oralpathogens.

Hydroxyapatite discs coated with saliva are suspended in a 1-litercontinuous flow reactor containing a growth medium appropriate for oralmicroorganisms (such as a dilute Nutrient Broth medium [0.05%]). Priorto beginning continuous flow, saliva (50 ml) is collected from humansubjects and added to the reactor. Microorganisms from the saliva areallowed to bind to the hydroxyapatite discs for one hour. Followingattachment, the discs are continuously washed with fresh medium for upto 5 days. In this manner, autoinducer will be continuously washed outand microorganisms will attach and grow on the hydroxyapatite discs.Following growth, microorganisms can be isolated on agar-containinggrowth medium; microorganisms can be identified by DNA sequencing asdescribed in Example 1.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific embodiments described herein. Such equivalents are consideredto be within the scope of this invention and are covered by thefollowing claims.

1.-17. (canceled)
 18. A method for isolating a novel bacterium in asample, comprising: (a) treating the sample with an effective amount ofan agent, wherein the agent interferes with, disrupts, removes, ordisenables a quorum sensing signal in the sample; (b) growing thetreated sample in a culture medium containing the agent; (c) isolatingcolonies of bacteria from the sample grown in step (b); and (d)analyzing the colonies to identify a bacterium having genetic noveltyminimally at the species or subspecies level.
 19. The method of claim18, wherein the agent inhibits Type 1 quorum sensing.
 20. The method ofclaim 19, wherein the agent is an enzyme that catalyzes a reaction withthe acyl homoserine lactone autoinducer.
 21. The method of claim 19,wherein the agent is selected from the group consisting of esterase,aminoacylase, carboxypeptidase, lipase, lactonase, peptidase andprotease.
 22. The method of claim 19, wherein the agent is a chemicalthat inhibits binding of the acyl homoserine lactone autoinducer to itsreceptor.
 23. The method of claim 19, wherein the agent is a chemicalthat inhibits biosynthesis of the acyl homoserine lactone autoinducer.24. The method of claim 18, wherein the agent inhibits Type 2 quorumsensing.
 25. The method of claim 24, wherein the agent is an enzyme thatcatalyzes a reaction with the Type 2 quorum sensing autoinducer.
 26. Themethod of claim 24, wherein the agent is a chemical that inhibitsbinding of the Type 2 quorum sensing autoinducer to its receptor. 27.The method of claim 24, wherein the agent is a chemical that inhibitsbiosynthesis of the Type 2 quorum sensing autoinducer.
 28. The method ofclaim 18, wherein the agent inhibits peptide regulated quorum sensing.29. The method of claim 28, wherein the agent is an enzyme thatcatalyzes a reaction with the peptide autoinducer.
 30. The method ofclaim 28, wherein the agent is selected from the group consisting ofpeptidase, deaminase and protease.
 31. The method of claim 28, whereinthe agent is a chemical that inhibits the binding of the peptideautoinducer to its receptor.
 32. The method of claim 28, wherein theagent is a chemical that inhibits the biosynthesis of the peptideautoinducer.
 33. The method of claim 28, wherein the agent is anantibody to the peptide autoinducer.
 34. The method of claim 18, whereinthe agent is a physical device for keeping the concentration of a quorumsensing autoinducer below a threshold concentration required for aquorum sensing response in the sample.
 35. The method of claim 18,wherein the sample is obtained from a human subject.